Considerable research has been conducted in recent years to develop improved means for the separation and collection of lymphocytes from human blood. An impetus for such research has been generated by the need for histocompatibility determinations in patients requiring organ transplants. A measure of lymphocyte function is critical to adjudge the type and level of medication necessary for immunosuppression.
One well-known method for isolating and collecting lymphocytes from anticoagulated human blood drawn via conventional phlebotomy techniques utilizes buoyant density centrifugation of blood cells. A newtonian fluid, frequently Ficoll-Paque.RTM., a liquid density gradient medium having a specific gravity of about 1.077 g/cc marketed by Pharmacia Fine Chemicals AB, Uppsala, Sweden, constitutes the medium. The method commonly involves the four general steps:
(a) a predetermined quantity of the Ficoll-Paque.RTM. medium is run into the bottom of a test tube;
(b) a sample of whole or diluted blood is carefully pipetted onto the medium;
(c) the test tube is placed in a centrifuge and the blood-medium combination centrifuged at about 400-500 G's for about 30-40 minutes to cause the components of the blood having specific gravities greater than the medium, viz. &gt;1.077 g/cc, to pass through the liquid; and thereafter,
(d) the lymphocytes, which have a specific gravity less than 1.077 g/cc, are pipetted off the medium.
Several problems or concerns have been found to be inherent in that technique. For example:
(1) if, during the pipetting of the blood sample into the separation medium, lymphocytes are inadvertently diffused below the surface of the medium, the specific gravity of the medium in that area is so reduced as to become inadequate to separate the lymphocytes;
(2) if, during centrifugation, lighter phases in the blood migrate into the separation medium, they cannot pass upward therethrough because the buoyant force generated by 400-500 G's is insufficient;
(3) centrifugation forces in excess of about 400-500 G's cannot be employed with Ficoll-Paque.RTM. medium as it is somewhat water soluble and higher centrifugation forces increase this solubility, thereby leading to a change in its specific gravity; and
(4) after centrifugation has been completed, the pipetting of the lymphocytes off the surface of the separation medium must be conducted with substantial care because of the newtonian character of the Ficoll-Paque.RTM. medium.
Numerous suggestions have been proposed for improving upon that technique. Several disclosures of such suggestions are recorded below.
U.S. Pat. No. 3,852,194 describes a process for isolating lighter phases from heavier fractions in human blood utilizing a thixotropic, gel-like material having a specific gravity which is intermediate to that of the phases to be separated. Upon centrifuging the gel and blood sample together, the gel exhibits sufficient flow to form a barrier between the lighter and heavier phases. That barrier enables the phase resting thereupon to be easily withdrawn therefrom using conventional laboratory techniques.
The patent postulates the operability of numerous gel-like substances; those substances complying with three general criteria:
(1) a specific gravity intermediate to that of the phases to be separated;
(2) chemical inertness to the phases of human blood; and
(3) essentially non-flowable when at rest (thixotropic).
U.S. Pat. No. 3,920,549 is asserted to comprise an improvement upon the disclosure of U.S. Pat. No. 3,852,194. That improvement involved the use of a solid element, termed an "energizer", having a specific gravity greater than that of the gel-like substance. This energizer, during centrifugation, impacts upon the gel, which is normally placed in the bottom of a blood collection tube, thereby expediting the upward movement of the gel along the walls of the tube. In this manner the energizer accelerates the isolation of the blood phases and permits a cleaner separation therebetween.
U.S. Pat. No. 4,190,535 is specifically drawn to a procedure for isolating lymphocytes, monocytes, and platelets from anticoagulated blood. The process contemplates three general steps:
(1) a water-insoluble, thixotropic gel-like substance having a specific gravity between about 1,065-1.077 g/cc and exhibiting chemical inertness to blood components is deposited into a sample of anticoagulated blood;
(2) the gel-blood combination is centrifuged at a force of at least 1200 G's for a sufficient length of time that the gel forms a barrier between the heavier blood cells and the lymphocytes, monocytes, and platelets; and then
(3) lymphocytes, monocytes, and platelets are removed from atop the barrier.
The patent observes that, because a non-newtonian, water-insoluble gel-like material capable of forming a barrier at centrifugation forces of in excess of 1200 G's is used, a faster and more complete separation was possible than with Ficoll-Paque.RTM. medium. The patent also observes that the elimination of the liquid density gradient medium avoids the time-consuming process of layering two liquids without mixing them.
U.S. application Ser. No. 528,401, filed Sept. 1, 1983 in the names of Richard J. Carroll, Albert A. Luderer, and Anthony R. Zine, Jr., and under the title of SEPARATION OF LYMPHOCYTES AND MONOCYTES FROM AGED BLOOD, is directed to improving the quality of the separation of lymphocytes and monocytes from aged samples of anticoagulated human blood by inhibiting the shift observed in the buoyant density of granulocyte white blood cells. The inventive process involves four general steps:
(1) a sample of anticoagulated blood is mixed with a hypertonic fluid containing an organic or inorganic ionic substance of relatively low molecular weight and which is chemically compatible with components of the blood;
(2) a water-insoluble thixotropic gel-like substance similar to that described in U.S. Pat. No. 4,190,535 with a specific gravity between 1.060-1.075 g/cc is deployed into the blood-hypertonic fluid mixture;
(3) the gel-blood-hypertonic fluid sample is centrifuged at a force of at least 1200 G's to cause the gel to form a barrier between the lymphocytes and monocytes and the heavier cells of the blood; and then
(4) the lymphocytes and monocytes are withdrawn from atop that barrier.
Whereas each of the above-discussed disclosures does indeed modify and improve upon various aspects of the well-known Ficoll-Paque.RTM. medium technique, none of them is able to equal or improve upon the performance of the liquid medium with respect to the purity of the separated cell population. Because purity is a critical parameter in cell separation, the above-discussed disclosures cannot be substituted for the Ficoll-Paque.RTM. medium technique in all applications. Consequently, research has continued in an effort to formulate simpler methods of cell separation which utilize a liquid medium. More particularly, a process has been sought which eliminates the time-consuming procedure necessary to layer blood samples onto the liquid density gradient medium without encountering mixing at the interface between the two liquids. This layering process generally requires about three minutes/tube to flow the blood sample down the inside wall of the tube at a rate which will permit layering and avoid turbulence at the interface. Inasmuch as this procedure is conducted manually and two tubes are conventionally prepared per sample, the setup time for readying a group of ten tubes may require a period of greater than one hour. The time involved in the centrifuging step is less critical since many tubes can be processed at the same time. Further simplification of the setup procedure could be accomplished if the patient's blood sample could be drawn directly into the centrifuge tube, thereby removing the need for transferring the sample form the collection tube to the centrifuge tube. In many instances it is desirable to add a reagent to the blood sample prior to cell separation to anticoagulate the blood, dilute the blood, or modify physical and/or chemical characteristics of the blood components.
Therefore, a primary objective of the present invention is to provide a series of devices which, separately or in combination, will not only satisfy the range of needs of research workers and diagnostic technicians who may merely wish to eliminate the layering problem or to minimize setup time, but also will provide a single product wherein all of the above-described benefits can be enjoyed.